Citation

  • Authors: Shao L. et al.
  • Year: 2022
  • Journal: Oncoimmunology 11 2010894
  • Applications: in vitro / in vivo / DNA, siRNA / in vivo-jetPEI, jetPRIME
  • Cell type: Hepa1-6

Method

in vitro: Hepa1-6 cells were seeded in 6-well plates overnight, then transfected with 1 μg pSBbi-luc plasmid and 1 μg Sleeping beauty SB100X transposase using the in vitro-jet PRIME transfection reagent according to the manufacturer’s instructions. in vivo: 1×10^7 Hepa1-6 cells or 1×10^7 Hepa1-6-GFP-luc+ cells were injected subcutaneously into the right flank of C57BL/6J mice. When tumors became palpable, mice were randomized into groups, which were intratumorally injected with either 1× phosphate-buffered saline (PBS), 20 μg scramble siRNA, 3p-scramble siRNA, GPC-3 siRNA, or 3p-GPC-3 siRNA, together with the in vivo-jet PEI transfection reagent (Polyplus Transfection Inc., New York, USA) every 3 days for a total of four doses. An anti-PD-1 antibody and a rat IgG2a isotype-matched control (RMP1-14, Bio X Cell, USA) were given intraperitoneally at a dose of 200 μg per mouse every 4 days for a total for four doses.

Abstract

Hepatocellular carcinoma (HCC) is associated with a high mortality rate and presents a major challenge for human health. Activation of multiple oncogenes has been reported to be strongly associated with the progression of HCC. Moreover, the immunosuppressive tumor microenvironment (TME) and the host immune system are also implicated in the development of malignant HCC tumors. Glypican-3 (GPC-3), a proteoglycan involved in the regulation of cell proliferation and apoptosis, is aberrantly expressed in HCC. We synthesized a short 5'-triphosphate (3p) RNA targeting GPC-3, 3p-GPC-3 siRNA, and found that it effectively inhibited subcutaneous HCC growth by raising type I IFN levels in tumor cells and serum and promoting tumor cell apoptosis. Moreover, 3p-GPC-3 siRNA was able to enhance the activation of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells while reducing the proportion of regulatory T cells (Tregs) in the TME. Most intriguingly, a blocking anti-PD-1 antibody improved the anti-tumor effect of 3p-GPC-3 siRNA, predominantly by activating the immune response, reversing immune exhaustion, and improving immune memory. Our study suggests that the combination of 3p-GPC-3 siRNA administration and PD-1 blockade may represent a promising therapeutic strategy for HCC.

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